Cloning, expression and purification of recombinant proteins
Full-length CHIPS1–121 and CHIPS with N-terminal truncation, composed of amino acids 31–121 (CHIPS ΔN) and CHIPS with N- and C-terminal truncations, composed of amino acids 31–113 (CHIPS ΔN/C) were cloned, expressed and purified as described previously . CHIPS with C-terminal truncation (CHIPS ΔC) and variants thereof were cloned in a modified pRSET B vector (Invitrogen, Carlsbad, CA). As a result of cloning, this protein was 112 amino acids long with two additional non-relevant amino acids included in the C-terminal end of the expressed protein. Cultivation was performed as described by Haas et al , but the protein was purified from inclusion bodies. Briefly, the CHIPS variants were purified from E. coli culture by lysis of the bacteria in 1/10 culture volume binding buffer (8 M urea, 20 mM Sodium Phosphate, 500 mM NaCl, pH 7.8). After sonication and filtration, the lysate was loaded onto a HisTrap Ni-column (GE Healthcare, Uppsala, Sweden). The column was washed with binding buffer, pH 7.8, pH 6.0 and pH 5.6, respectively. CHIPS was eluted with binding buffer, pH 5.6 with 50 mM EDTA and fractions containing protein as measured by A280 were pooled and refolded by dialysis against 50 mM Tris-HCl, pH 8.0. The His-tag was removed as described .
Site-directed mutagenesis and expression of CHIPS variants in plate format
To be able to compare many mutants of truncated CHIPS, a tag is required to capture the proteins and determine relative concentrations. However, CHIPS activity has proven to be sensitive to the addition of His-tags in both the N- and C-terminus. These have to be cleaved off before functional assays can be performed. Instead, we used a mAb (2H7) recognition site located between residue 15 and 30 within the native CHIPS N-terminus that does not interfere with C5aR blocking activity . Therefore the mutations were generated in CHIPS with C-terminal truncation, leaving the N-terminal 30 amino acids untouched for recognition by the 2H7 mAb in ELISA. Site-directed mutagenesis was performed in order to introduce point mutations into the CHIPS sequence with C-terminal truncation. This was performed using the QuikChange II mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's recommendations. The new CHIPS variants were sequence verified and transformed into E. coli BL-21 Star(DE3)pLysS (Invitrogen, Carlsbad, CA) for protein expression. Variants of CHIPS with C-terminal truncation were expressed in LB medium containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol in deep-well plates. Overnight cultures were diluted 1/50 in fresh LB containing 50 μg/ml ampicillin and cultured at 37°C for 3 hours. IPTG (Isopropyl β-D-Thiogalactoside) (final concentration 0.5 mM) (BDH, Poole, UK) was added to log-phase cultures to induce protein expression followed by cultivation for three hours. Protein was prepared from E. coli lysates by freeze-thawing the E. coli pellet in a buffer consisting of PBS-0.05% Tween-20, Complete EDTA-free protease inhibitor (Roche, Basel, Switzerland), 25 U/ml Benzonase (Sigma-Aldrich, St Louis, MO) and 1 KU/ml rLysozyme (EMD Chemicals, Darmstadt, Germany). The lysates were incubated for 30 min at room temperature with shaking and centrifuged at 13,000 rpm for 15 min to remove debris and were stored at -20°C.
Affinity purification of anti-peptide IgG and anti-CHIPS IgG
A peptide, comprising the phage derived sequence MNKSYTI, was synthesized with an additional C-terminal spacer of three glycines and a cysteine for efficient coupling. (Bio-synthesis; Lewisville TX). A 38-mer peptide comprising the N-terminal part of CHIPS (first 37 amino acids plus an additional cysteine (Pepscan, Lelystad, The Netherlands) served as control. In order to couple the peptides to a solid matrix, peptides were first reduced using agarose linked Tris(2-Carboxyethyl) Phosphine (TCEP) (Pierce, Rockford, IL) and subsequently mixed with Sulfo-Link agarose beads (Pierce, Rockford, IL) in 50 mM Tris/HCl, pH 8,3 with 5 mM EDTA for 2 hours at room temperature. Unreacted groups were blocked with L-cysteine and beads were extensively washed with coupling buffer and PBS. Small 1 ml columns were used for affinity purification of IgG from a pooled human immunoglobulin preparation for intravenous use (IV-IgG) (Sanquin, Amsterdam, The Netherlands). IgG was eluted with 0.1 M glycine-HCl buffer pH 2.9 and neutralized with 1 M Tris pH 8.0. Pooled fractions were dialyzed overnight against PBS and the purified anti-peptide IgG was stored at 4°C.
Purified CHIPS variants (full-length or CHIPS ΔN/C) were coupled to CNBr activated sepharose 4B (Amersham Biosciences, Uppsala, Sweden) and packed on a column according to manufacturer's instructions. Affinity purification was performed on an ΔKTA Prime system (Amersham Biosciences) according to the manufacturer's protocol. A total of 1 g IV-IgG (20 mg/ml) over the column. Bound human IgG was eluted with 0.1 M Glycine pH 3.0 and the pH neutralized with 1 M Tris, pH 8.0. Eluted fractions containing protein were pooled and buffer was changed to PBS on PD-10 columns (Amersham Biosciences). The affinity-purified human anti-CHIPS IgG was stored at 4°C.
ELISA was used for many applications throughout the study. Nunc Maxisorb clear or white 96 well plates were coated with the specific protein in PBS. Incubations were carried out in a volume of 50 μl for 1 hour at RT of not described differently, always followed by washing trice with PBS-0.05%Tween-20. Dilution buffer for all reagents was PBS-0.05%Tween-20 with 1% BSA or milk powder added.
Anti-CHIPS and Pepscan ELISA
Microtiter plates were coated with CHIPS (1 μg/ml) overnight at 4°C. Plates were blocked with PBS-0.05%Tween-20 4% BSA and incubated with different anti-CHIPS antibodies in dilution buffer with 1% BSA. Bound antibodies were detected with goat-α-human-IgG conjugated with peroxidase (Southern, Birmingham, USA) and TMB as substrate. For peptide experiments, synthetic 25-mer CHIPS derived peptides spanning the complete CHIPS1–121 sequence were synthesized as described earlier . Plates were coated with peptide (10 μM) overnight at 4°C and ELISA was performed as described above with IgG purified on full-length CHIPS1–121.
Binding of human anti-CHIPS ΔN/C IgG to CHIPS variants with C-terminal truncation
For quantification of expressed proteins, plates were coated overnight at 4°C with 3 μg/ml monoclonal anti-CHIPS Ab 2H7 directed against a peptide of CHIPS amino acids 24–30 . Plates were blocked in PBS-0.05% Tween-20 with 3% milk powder, washed and incubated with serial dilutions of lysates from CHIPS variants in dilution buffer with 1% milk powder. Binding was detected with 3 μg/ml polyclonal rabbit anti-CHIPS N-terminal IgG (IgG produced by immunization of a rabbit with a KLH-coupled synthetic peptide corresponding to CHIPS N-terminal amino acids 1–14) and goat anti-rabbit IgG-HRP (Southern Biotech, Birmingham, AL)). Super Signal ELISA Pico Chemiluminescent Substrate (Pierce, Rockford, IL) was added and luminescence was measured.
For detection of binding of human anti-CHIPS IgG to the CHIPS variants, plates were coated, blocked and incubated with E. coli lysates as described above. Affinity-purified human anti-CHIPS ΔN/C IgG was added and binding was detected with goat-anti-human IgG HRP (Jackson ImmunoResearch, West Grove, PA) and substrate as described above.
Random peptide phage libraries and phage selection
The Ph.D.-7™ and Ph.D.-C7C™ libraries from New England Biolabs (Ipswich, MA) were used to map the epitopes for human IgG on the surface of the CHIPS protein.
The Ph.D.-7™ Phage Display Peptide library consists of 7-mer random linear peptides fused with a linker sequence (GGGS) to the N-terminus of the major coat protein pIII of bacteriophage M13. The randomized segment of the Ph.D.-C7C™ library is flanked by a pair of cysteine residues, which are oxidized during phage assembly to a disulfide linkage, resulting in the displayed peptides being presented to the target as loops . Protein G coated beads (100 μl) (Dynal, Norway) were washed three times with 1 ml PBS-0.05% Tween-20. The washed beads were blocked with 1 ml PBS-0.05% Tween-20, 5% BSA for 1 h at 22°C. Beads were washed four times and resuspended in 1 ml PBS-0.05% Tween-20. One half of the blocked beads was used for preclearing the phage stock. Therefore, 10 μl Ph.D.-7™ and 10 μl Ph.D.-C7C™ were incubated with blocked beads in PBS-0.05% Tween-20 and were incubated for 30 min at 22°C under continuous agitation. Affinity-purified human anti-CHIPS ΔN/C IgG (final concentration ~10 nM) was added to the precleared phages and incubated at 22°C for 30 min. The phage/IgG suspension was added to the remaining blocked beads and incubated at 22°C for 30 min. The beads were washed 10 times with PBS-0.05% Tween-20 to wash away unbound phages. The bound phages were eluted with 0.2 M Glycine, pH 2.2, 0.1% BSA for 8 min followed by neutralization of the pH with 1 M Tris-HCl, pH 8. The eluate was amplified and 10 μl of amplified phages was used as input for the next selection round. To increase the specificity of the phage selection, the bound phages in the fourth round were eluted using competition elution with the CHIPS protein. Bound phages were eluted by overnight incubation with 1.8 mg/ml CHIPS.
Phage titration, amplification and characterization
LB medium was inoculated with a single colony of ER2738 E. coli and incubated at 37°C with vigorous shaking until mid-log phase (OD600 ~0.5). Top agar (50% LB agar, 50% LB medium) was melted and cooled to approximately 45°C. Melted top agar (3 ml) was added to 200 μl ER2738 E. coli and poured on top of a LB/0.5 mM IPTG/80 μg/ml X-gal plate. 10 μl of 10-fold dilutions in LB medium was spotted on the prepared culture plates and incubated overnight at 37°C. The next day, plaques were counted in order to calculate phage titers. The remaining phage eluate was added to ER2738 E. coli culture at early log phase (OD600 0.4–0.5) and incubated with vigorous shaking at 37°C for 4.5 h. Cultured cells were pelleted at 4°C and phages were precipitated overnight at 4°C in 25% PEG6000 (Sigma-Aldrich, St Louis, MO) 3 M NaCl. The precipitated phages were centrifuged for 15 min at 10,000 rpm, 4°C. The pellet containing the amplified phages was resuspended in 200 μl PBS and titrated as described above. After the fourth selection round no phage amplification was performed and phages were directly characterized by DNA sequencing.
48 different plaques from the titration plates were stabbed with a pipette tip and used to infect 1 ml 1/100 diluted overnight culture of ER2738 E. coli and incubated for 4.5~5 h at 37°C. Cultures were centrifuged and 500 μl of the supernatant was precipitated with PEG6000, 3 M NaCl for 10 min at 22°C. The samples were centrifuged for 10 min at 13,600 rpm and the pellet was resuspended in 100 μl 4 M NaI, 10 mM EDTA, pH 8. 250 μl 95% EtOH was added and the samples were incubated for 10 min at 22°C to preferentially precipitate the single-stranded phage DNA. Samples were centrifuged for 10 min at 13,600 rpm and the pellet was washed with 70% EtOH, dried and sent for sequencing to MWG Biotech (Martinsried, Germany) using the PIII-96seq primer (New England Biolabs, Ipswich, MA).
A phage ELISA was performed to test the binding specificity of the selected phages for affinity-purified human anti-CHIPS ΔN/C IgG. Affinity-purified human anti-CHIPS ΔN/C IgG was coated overnight with at 4°C. Coated and non-coated wells were blocked with PBS-0.05% Tween-20, 5% BSA. Serial dilutions of the purified phage stocks in dilution buffer with 1% BSA were added and incubated for 1 h at 37°C. Detection was performed with mouse anti-M13-mAb (1 μg/ml) (Amersham Biosciences, Uppsala, Sweden) and rabbit anti-mouse IgG-HRP (Dako A/S, Glostrup, Denmark) and OPD substrate (O-phenylenediamine) (Sigma Aldrich, St Louis, MO).
Analysis of peptide antibody binding to CHIPS
Binding of affinity-purified antibodies and pooled human IgG (IV-IgG) to CHIPS was studied by ELISA as described for anti-CHIPS ΔN/C antibodies and by SPR on a Biacore 1000 instrument. 20 μl 1 mg/ml CHIPS was directly coupled to an activated N-ethyl-N'(di-methylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) carboxymethyl dextran sensor chip (CM5). Unreacted groups were blocked by injection of 50 μl 1 M ethanolamine-HCl pH 8.5. Binding assays were performed at a constant flow rate of 5 μl/min at 25°C. Affinity-purified antibodies and IV-IgG were diluted in HBS-EP buffer (10 mM HEPES (pH 7.4) containing 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20). Antibodies were allowed to interact with CHIPS for 210 s followed by a 120 s dissociation phase. Additionally, the antibodies were preincubated with 1 mg/ml CHIPS protein to study competition. Affinity-purified anti-peptide antibodies were tested at a concentration of 10 μg/ml. Residual bound antibody was removed from the sensor chip surface by washing the chip for three minutes with 10 mM glycine-HCl (pH 1.5).
The amino acid sequences of the selected phages were aligned using the Clustal-W alignment tool http://www.ebi.ac.uk/clustalw/. Consensus sequences were manually mapped onto the surface of the CHIPS protein using the CHIPS31–121 NMR structure (PDB code: 1XEE)  and the PyMOL molecular graphics system .
Biological activity assay
The C5aR inhibiting capacity of the purified CHIPS variants with C-terminal truncation was tested by measuring calcium release from C5a stimulated U937 cells stably transfected with the C5aR . U937 cells (human promonocytic cell line) transfected with the C5aR (U937/C5aR) were a generous gift from Dr. E. Prossnitz (University of New Mexico, Albuquerque, NM). Cells were grown in 75 cm2 cell culture flasks in a 5% CO2 incubator at 37°C and were maintained in RPMI 1640 medium with L-glutamine (Cambrex, Verviers, Belgium) and 10% FBS (Cambrex, Verviers, Belgium). Briefly, 5 × 106/ml U937/C5aR cells were incubated with 2 μM Fluo-3AM (Sigma Aldrich, St Louis, MO) in RPMI 1640 medium with 0.05% BSA for 30 min at room temperature. After washing, the cells were preincubated with buffer or a 3-fold dilution series of CHIPS variants (1 to 0.003 μg/ml) at room temperature for 30 min. Every sample was measured on a FACSsort flow cytometer (BD Biosciences, San José, CA) and C5a (Sigma Aldrich, St Louis, MO) (final concentration 1 nM) was added. The cell population was gated on forward and side scatter and results are expressed as percentage inhibition of calcium release as compared to cells without addition of CHIPS.
The structures of the purified C-terminally truncated CHIPS variants were compared by the use of CD spectroscopy. The experiment was carried out on a Jasco J-720 spectropolarimeter (Jasco Inc., Easton, MD) in a 2 mm cuvette at a protein concentration of ~20 μM. Spectra were recorded from 195 to 250 nm at 20°C, at a scan speed 50 nm/min, a time constant of 4 s, a bandwidth of 1 nm, resolution of 1 nm and sensitivity of 50 mdeg.
Data from ELISA and cellular experiments were plotted by the use of the Prism 4 software package (GraphPad Software Inc.). The cellular data were fitted in a nonlinear regression model (sigmoidal dose-response curve with variable slope).